CN112023062A

CN112023062A - Method for inhibiting allergic reactions using soluble IgE receptors

Info

Publication number: CN112023062A
Application number: CN202010957975.9A
Authority: CN
Inventors: 不公告发明人
Original assignee: Beijing Gene An Technology Co ltd
Current assignee: Beijing Gene An Technology Co ltd
Priority date: 2020-09-18
Filing date: 2020-09-18
Publication date: 2020-12-04

Abstract

IgE binds to IgE receptors on the cell surface of mast cells and the like, and is a key link for inducing allergic reactions of tissues such as nasal mucosa and the like. The present invention provides a method of inhibiting allergic reactions by inhibiting the binding of IgE to mast cell IgE receptors. The method mainly comprises the following steps: preparing mRNA encoding FCERI-ECD in vitro, wherein the mRNA comprises a stop codon, a 5-terminal translation region sequence and a 3-terminal translation region sequence; packaging FCERI-ECD-mRNA with liposome; the packaged FCERI-ECD-mRNA is administered locally (e.g., nasal mucosa, skin, etc.) or systemically (e.g., vein) to the human body. FCERI-ECD mRNA expresses FCERI-ECD protein in vivo, binds and inhibits IgE, and prevents IgE from binding IgE receptors on cell surfaces such as mast cells, thereby inhibiting anaphylaxis.

Description

Method for inhibiting allergic reactions using soluble IgE receptors

Technical Field

The invention belongs to the field of biological medicine, and provides a novel method for inhibiting anaphylactic reaction by using macromolecular RNA.

Background

Under normal conditions, after entering the human body, foreign substances are mostly exposed to two fates, and if recognized as useful or harmless substances by the body, the substances are harmonious with the human body and finally absorbed, utilized or naturally discharged. If these substances are identified as harmful substances, the immune system of the body reacts immediately to expel or destroy them, which is the protective effect exerted by the immune response. The immune response is one of the important functions of the human defense system, but if the response is outside the normal range, i.e. the immune system attacks harmless substances, this situation is called an allergy. Allergy is a disease, because the healthy body tissues are damaged by the endless attack, and even the immune system attacks and destroys the body tissues naturally and sometimes, which is very bad for the health of the human body.

Allergies are classified as type I (immediate), type ii (cytotoxic), type iii (immunocomplex), type iv (delayed). Anaphylaxis is representative of type I allergy. The common allergic diseases include allergic asthma, allergic rhinitis, pollinosis, dermatitis, etc. For example, allergic rhinitis is mostly the result of interaction between inhaled allergens and nasal mucosa of patients, and patients are mostly allergic in constitution and have heredity.

The allergic rhinitis is clinically classified into seasonal rhinitis and perennial rhinitis, the former is caused by pollen inhalation, and the latter is caused by dust, mites, molds, animal hair, dander, bird feather and the like in the house. The clinical manifestations are as follows: sudden rhinocnesmus, continuous sneezing (more than 5) and a large amount of clear watery serous nasal secretion, the symptoms of each outbreak usually last for more than 1 hour, and the outbreak is repeated.

Allergic asthma, which is the main type of bronchial asthma, is a respiratory tract obstruction syndrome characterized by reversible airway and spastic stenosis caused by a wide airway hypersensitive state caused by an allergen or other allergic factors, and the pathological changes mainly affect the bronchi. The main causes of the outbreak are inhalant allergens (indoor dust, house dust mites and dust mites, but also fungal spores, various plant pollens, animal dander, feathers, silk, old fabrics, insect limbs, debris, feces, molting, insect eggs, etc.), food (such as certain eggs, etc.), drugs (aspirin, etc.).

The existing commonly used anti-allergic drugs comprise antihistamine and dexamethasone hormone, and have great side effects. Antihistamines can have side effects such as headache, dry mouth, drowsiness, and if severe, can induce glaucoma, leading to rare, severe cardiotoxicity, and fatal arrhythmia. The long-term use of dexamethasone hormones can cause the following side effects: iatrogenic cushing syndrome facial and body condition, weight gain, lower limb edema, purple marks, tendency to bleed, poor wound healing, acne, menstrual disorders, ischemic necrosis of the humeral or femoral head, osteoporosis and fractures (including spinal compression fractures, long bone pathological fractures), muscle weakness, muscular atrophy, hypokalemic syndrome, gastrointestinal irritation (nausea, vomiting), pancreatitis, peptic ulcers or perforations, inhibited growth in children, glaucoma, cataract, benign intracranial pressure elevation syndrome, impaired glucose tolerance and exacerbations of diabetes.

In conclusion, there is an urgent need to develop new methods for suppressing allergic reactions.

Mast cells are mainly present in mucosal and connective tissues and, by virtue of the cross-linking of the high affinity receptor FCERI of IgE expressed on their surface with the anti-allergen specific antibody IgE, form an IgE/FCERI complex, which in turn triggers a type I allergy (fig. 1). The mast cell plasma contains a large number of granules in which biological mediators such as histamine are stored, and activated mast cells can also newly synthesize biological mediators such as TNF-alpha, IL-6 and IL-13. It is with these bioactive mediators that mast cells exert their biological effects during allergic diseases. Therefore, the expression of the mast cell surface receptor FcRI and the biological function regulation mechanism thereof provide a theoretical basis for the prevention and treatment of allergy. In acute allergic reactions, specific IgE binds to the surface of tissue mast cells, and the generation of antigen-dependent mast cell activation is a central event and key element of allergic reactions. IgE is the lowest concentration of immunoglobulin subtype in the blood circulation. In some individuals with allergic disease or parasitic infections, its plasma concentration can be significantly elevated. When the IgE bound with the antigen is simultaneously bound with FCERI and the like on the surface of mast cells, FCERI is activated to release mediators from the mast cells and other effector cells, and allergic and inflammatory reactions are caused.

FCERI is a receptor on the surface of mast cells and belongs to a transmembrane protein. The extracellular phase of FCERI (FCERI-ECD) is the site of IgE binding (fig. 2), while the intracellular phase has the function of transmitting signals into the cell, inducing mast cell activation and degranulation. Our strategy is to allow tissues in vivo to synthesize the extracellular domain of FCERI (FCERI-ECD) FCERI-ECD which, upon binding to IgE in vivo, prevents IgE binding to endogenous transmembrane FCERI, preventing intracellular signal transduction of FCERI, thereby inhibiting mast cell activation and allergic reactions (fig. 3).

Disclosure of Invention

The present invention provides a method of inhibiting allergic reactions by inhibiting the binding of IgE to the IgE receptor of mast cells. The method mainly comprises the following steps: preparing in vitro an mRNA encoding FCERI-ECD comprising adding a stop codon, a 5 'UTR (untranslated region) and a 3' UTR sequence to the ORF (open reading frame) of FCERI-ECD; packaging FCERI-ECDMNA with liposome; the packaged mRNA is administered topically (e.g., nasal mucosa, skin, etc.) or systemically (e.g., intravenously) to the human body.

We note that publication No. CN1922197A (published: 2007-2-28) discloses a method for targeted delivery of RNA interference molecules for the treatment of IgE-mediated disorders, comprising preparing a genetic construct of siRNA targeting IgE, the target gene being selected from IgE, FcRI, etc. Our invention is different from this patent. The patent is a treatment with a DNA vector, and our invention is the use of mRNA; the patent uses a DNA expression vector to express siRNA, the siRNA is internationally recognized small molecular RNA, and the siRNA uses macromolecular RNA; the aim of this patent is to knock down the expression of both IgE and IgE receptors, and our invention is primarily to use mRNA to highly express the extracellular domain of IgE receptor in vivo, allowing it to bind IgE, thereby preventing IgE binding to IgE receptors in vivo.

The invention provides a novel method for inhibiting anaphylactic reaction besides the common compounds such as antihistamine and hormone.

Drawings

FIG. 1 the mechanism by which IgE participates in the development of allergic reactions.

FIG. 2 Structure of the IgE receptor FCERI. The extracellular phase within the circle (FCERI-ECD) is the IgE binding domain.

FIG. 3 strategy for the use of FCERI extracellular domain (FCERI-ECD) mRNA.

FIG. 4 comparison of nasal scrape times of mice in the placebo group and the allergic rhinitis model group.

FIG. 5 entry of FCERI-ECD mRNA through mucosa. After the fluorescence labeled mRNA is locally sprayed through a nasal cavity, the tissue fluorescence value of the nasal mucosa of the mouse changes. The control mean was 0.94. + -. 0.15 and the FCERI-ECD mRNA group mean was 30.6. + -. 6.

Detailed Description

Firstly, preparing blank liposome:

1. experimental materials:

soy lecithin, cholesterol (alatin); DOTAP (Toronto Research Chemicals, Canada);

dichloromethane (modern oriental); 0.22um polycarbonate fiber film (Ireland)

2. The main apparatus is as follows:

rotary evaporator (great wall instruments, china); an electric heating constant temperature water bath (Shanghai essence hong); misonix XL-2000S cell sonicator (Misonix corporation, usa); electronic balances (Sartorius, germany); microsyrinths (Ependorf, germany).

3. Liposome preparation

(1) Precisely weighing 6mg of soybean phospholipid, 6mg of DOTAP and 3mg of cholesterol, mixing and dissolving in 5mL of dichloromethane, and carrying out rotary evaporation in a water bath at 28 ℃ to remove the solvent;

(2) adding 3mL of distilled water preheated to the same temperature into a water bath at 50 ℃, and hydrating for 15min to obtain liposome suspension;

(3) performing ultrasonic treatment (100w, 1min) on liposome suspension with ultrasonic cell pulverizer, and filtering with 0.22 μm polycarbonate membrane to integrate liposome microparticles to obtain the desired liposome.

II, mRNA preparation

First, a double-stranded DNA template with a T7 promoter was synthesized, encoding mouse, human FCERI-ECD and GFP, respectively. We added UAG as a stop codon for protein translation after addition of the Open Reading Frame (ORF) encoding FCERI-ECD. We added the 5 'UTR region of pcDNA3 to the front of the ORF and the 3' UTR and polyadenylation regions of bPA to the back of the ORF. The purpose of these treatments is to enable efficient translation of mRNA into protein in vivo. We prepared mRNA in vitro using T7 polymerase mediated DNA-dependent RNA transcription.

1. In a test tube, the following reagents were added at room temperature in one portion:

5x transcription buffer 100ul

DTT(100mM)100ul

RNase inhibitor 500 unit

rNTP capping mix(see Section II)100μl

Ribo m 7G Cap Analog,5mM 25μl

DNA template (10ug)10ul

T7 RNA polymerase 200 units

Add ddH2O to 500ul

And (5) culturing at room temperature for 3 h.

RNA purification:

(1) to 180. mu.l of the reaction product was added 20. mu.l of 3M sodium acetate, pH 5.2, and mixed thoroughly.

(2) An equal amount of phenol/chloroform mixture was added, followed by extraction twice with chloroform. The aqueous phase was collected and transferred to a new tube.

(3) RNA was precipitated by adding 2 volumes of ethanol. Incubate at-20 ℃ for at least 30 minutes and centrifuge to collect the pellet.

(4) The supernatant was removed and the pellet was washed with 500. mu.l of cooled 70% ethanol.

(5) Drying the precipitate, adding acetic acid buffer solution, and freezing for storage.

Preparation of mRNA-liposome complexes

(1) 250 μ l of mRNA (1 μ M) was added dropwise to 250 μ l of blank cationic liposome suspension to obtain a mixture having a mass ratio [ DOTAP in liposomes, i.e., M (DOTAP): mRNA) ] of 10: 1.

(2) Vortex for 10min, incubate at room temperature for 30min to form mRNA-liposome complex.

Fourth, model establishment of allergic rhinitis

1. Animals:

40 SPF female BALB/c mice of 7 weeks old were kept in the barrier layer of the animal, at room temperature of 20-24 deg.C for 12h/d of illumination time, and were fed freely (kept in the barrier environment for 1 week, excluding environmental factors from affecting the experimental results).

2. The main reagents are as follows:

ovalbumin (pekoran novi, beijing); alum (rainbow lake chemical); PBS buffer (sequoia cuneata bridge); sodium chloride (Beijing chemical plant).

Preparation of OVA solution and preparation of allergic rhinitis animal model

Nasal itching and sneezing are characteristic external clinical manifestations of allergic rhinitis, a type i allergic reaction characterized by mast cell infiltration. Ovalbumin is powdery and has strong immunogenicity, so the ovalbumin solution is selected as the allergen of the experiment.

We tried 6 different mouse rhinitis models and found that animal models can be successfully prepared within 30 days by three-stage treatments of sensitization, reinforcement and activation with ovalbumin. The animal model is proved to be successful by the times of grabbing the nose of the animal, infiltration of mast cells under nasal mucosa and the like.

And (3) sensitization stage:

(1) weighing 10 μ g of ovalbumin and 2mg of alum respectively by using an electronic balance;

(2) preparing a PBS solution with pH of 7.3 according to a preparation method of the PBS buffer solution;

(3) dissolving the weighed ovalbumin and the weighed alum in 0.5mL PBS to prepare a solution of 20 mu g of ovalbumin and 4mg of alum per milliliter;

(4) a solution of 10. mu.g ovalbumin and 2mg alum in 0.5mL PBS was injected intraperitoneally once a day, for a total of 7 times, for a total of 13 days. And then enters the strengthening stage.

And (3) strengthening stage:

(1) accurately weighing 0.5g of ovalbumin by using an electronic balance;

(2) dissolving the weighed ovalbumin in 50mL PBS to prepare a solution of the ovalbumin with the concentration of 10mg/mL, wherein the solution is used in the strengthening stage of the mice;

(3) from day 14 onwards, a liquid containing 1% ovalbumin (10mg/mL in PBS) was tested in spray form for 5 consecutive days at 30min per day;

an activation stage:

(1) weighing 40mg of ovalbumin by using an electronic balance, dissolving the ovalbumin in 1mL of 0.9% NaCl solution to prepare an ovalbumin solution with the concentration of 4%, wherein the solution is used for activating the mice;

(2) after 3 days of rest, 2% ovalbumin was instilled into the bilateral nasal cavities with a micropipette, 20 μ l per side, once a day, 7 consecutive times. Within 30min after administration, the behavioral changes of the mice were observed and recorded;

(3) after 2 days, the number of nasal bites per 30min of the mice was recorded and samples of the mice were collected.

Successful preparation confirmation of mouse allergic rhinitis model:

(1) the number of nasal grabbing times:

nasal itching and sneezing are characteristic external clinical manifestations of allergic rhinitis. The number of nasal graspings per 30min of the mice was determined. The number of nasal paw picks was significantly increased in the model group mice (fig. 4).

(2) Mast cells were assayed with toluidine blue:

allergic rhinitis is a type i allergic reaction characterized by mast cell infiltration. Toluidine blue is one of the commonly used synthetic dyes, belonging to the quinone imine dyes, which generally contains two chromophores, one of which is an amine group and the other is a quinoid benzene ring, to form chromogen coloration. In addition to chromophores, dyes also have groups of atoms that provide the chromogen with affinity for tissues and other substrates, i.e., color enhancement. The chromophor can promote the dye to generate ionization into salt, and the chromophor can generate dyeing power to the tissue to color the tissue cells on the section. Toluidine blue contains not only two chromophores, but also two auxochromes, and is a basic dye, and cations in toluidine blue have a dyeing effect, and acidic substances of tissue cells are combined with the cations to be dyed. Staining cell nuclei to make them blue; the mast cell cytoplasm containing metachromatic substances such as heparin, histamine and the like can be in metachromatic purplish red when encountering toluidine blue, so the toluidine blue can be used for detecting the mast cell.

Toluidine blue is a mast cell specific stain. And (3) dyeing results: mast cell granules appear purplish red and the nucleus appears blue. Mast cell cells in the blank group were approximately round and less numerous; mast cells in the experimental group appeared irregular in shape and purple-red granules were seen, a degranulation phenomenon in the case of sensitization of mast cells. Furthermore, the change of the number and the shape of the mast cells after the model forming of the allergic rhinitis is successful is determined.

(3) Immunohistochemistry was used to determine infiltration of mast cells under the nasal mucosa:

mast cells are mainly present in mucosal and connective tissues and, by virtue of the cross-linking of the high affinity receptor FcRI of IgE expressed on their surface with the anti-allergen specific antibody IgE, form an IgE/FcRI complex, which in turn triggers a type I allergy. We performed immunohistochemical staining with anti-FcRI antibody, confirming that the model group expresses a large number of mast cells.

4. Experiment grouping

40 mice were randomly divided into 4 groups of 10 mice each.

(1) Blank control group:

PBS is used to replace the PBS solution containing ovalbumin to treat the mice in the stages of sensitization, strengthening and activation. The number of nasal paw picks was recorded daily for 14 consecutive days from day 16; the number of nasal paw counts per 30min of mice was recorded at day 30 and all mouse samples were collected. Mice did not develop allergic rhinitis-like symptoms.

(2) Model group:

firstly, ovalbumin is used for making a mouse allergic rhinitis model;

(vii) from day 16 onwards, spraying PBS once daily to the nasal cavity;

recording the number of times of grabbing the nose of the mouse every day; all mouse samples were collected at day 30.

(3) Model negative control group:

firstly, ovalbumin is used for making a mouse allergic rhinitis model;

② from day 16, spraying liposome-encapsulated control mRNA (GFP mRNA) to nasal cavity once a day;

(4) Model experimental treatment group:

firstly, ovalbumin is used for making a mouse allergic rhinitis model;

② from 16 days, the nasal cavity is sprayed with liposome-wrapped mRNA (FCERI-ECD mRNA) (containing 2ug of mRNA) once a day;

Fifth, experimental results

Topical mRNA dosing confirmation:

after topical spray treatment of the nose of the animals with fluorescently labeled mRNA and sectioning of the nasal tissue, fluorescence measurements showed that mRNA could enter the nasal tissue (fig. 5).

2. The number of nasal grabbing times:

the number of nasal griping times per 30min for the mice of the blank control group was about 1 on average; the number of nasal grabbing times of animals of the animal nasal anaphylactic reaction model group made of the ovalbumin can reach 40 times per 30 min. There was no significant improvement in nasal grab frequency after treatment with GFP mRNA from the negative control group, but the nasal grab frequency was significantly reduced in animals treated with FCERI-ECD mRNA (table 1). The FCERI-ECD mRNA is suggested to have the effect of inhibiting anaphylactic reaction.

TABLE 1 Effect of soluble IgE receptor mRNA on nasal scrape frequency of allergic rhinitis in mice

Group (n is 6)	Number of nasal graspers (every 30min)
		Normal control group	1.00±0.31
Model set	42.56±1.40
		Model yin-shape therapeutic group	41.32±2.31
Model experiment treatment group	12.65±2.23^****

^****P<0.0001, compared to negative treatment control (GFP mRNA).

3. Mast cell infiltration:

we performed mast cell staining of nasal mucosal tissue in normal mice, allergy model group, negative treatment combination model experimental treatment group with toluidine blue, and the results are shown in table 2. Mast cell staining was significantly less in the FCERI-ECD mRNA treated group compared to the model combination negative treated group, suggesting that treatment with FCERI-ECD mRNA inhibited allergic reactions. Receptors for IgE antibodies were highly expressed in mast cells and we performed immunohistochemical staining of these tissues with anti-FCERI antibodies (anti-intracellular segment) showing that FCERI-ECD mRNA treatment reduced the number of mast cells.

TABLE 2 mast cell infiltration assay

In conclusion, animal models demonstrate that FCERI-ECD mRNA has a significant inhibitory effect on allergic reactions.

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Claims

1. An antiallergic agent which inhibits the activity of IgE characterized by a macromolecular RNA encoding an extracellular domain of an IgE receptor.

2. A macromolecular RNA according to claim, which is produced by transcription with RNA polymerase using DNA as a template.

3. The allergic disease to which the antiallergic agent according to claim 1 is directed, which comprises at least one of allergic rhinitis, vernal mucositis, atopic dermatitis, allergic conjunctivitis, nettle diagnosis, and bronchial asthma.